Difference between revisions of "Team:Heidelberg/Protein Interaction"
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As all PREDCEL/PACE experiments are based on phage infection, selection for efficient T7-RNAP auto-reassembly starts with a phage injecting its genome, the selection plasmid (SP), into a bacterial cell. The SP carries the two halves of the split T7 RNAP expressed under one geneIII (gIII) promotor. Further it contains required genes for phage reproduction except for gIII. | As all PREDCEL/PACE experiments are based on phage infection, selection for efficient T7-RNAP auto-reassembly starts with a phage injecting its genome, the selection plasmid (SP), into a bacterial cell. The SP carries the two halves of the split T7 RNAP expressed under one geneIII (gIII) promotor. Further it contains required genes for phage reproduction except for gIII. | ||
If through MP-activation mutation process leads to beneficial mutations in one or both T7 RNAP sequences, split variants assemble efficiently, bind to T7 promotor and thereby activate expression of protein III (pIII). Now phages can be built including pIII. That enables the phage to be released and to infect new cells (left). | If through MP-activation mutation process leads to beneficial mutations in one or both T7 RNAP sequences, split variants assemble efficiently, bind to T7 promotor and thereby activate expression of protein III (pIII). Now phages can be built including pIII. That enables the phage to be released and to infect new cells (left). | ||
| − | If mutations are inefficient, T7 RNAP halves do not reassemble, therefore cannot bind to T7 promotor and | + | If mutations are inefficient, T7 RNAP halves do not reassemble, therefore cannot bind to T7 promotor and pIII is not expressed. Therefore, phages cannot leave cells or infect new cells (right).| |
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Revision as of 15:03, 1 November 2017
Protein Interaction
Improving Split Protein Auto-Reassembly
Introduction
Protein Interactions in Evolution Context
The proteome reflects the entirety of proteins that is expressed by a cell or an organism. Most of these proteins do not function alone. Instead, they are dependent on the interactions with a multitude of other proteins to fulfill their functions within the cell. Protein-protein interactions are responsible for almost any kind of cellular processes, such as signal transduction, cell-cell contact, transport, metabolism, cell motion or even antigen-antibody recognition (Larance, Nature Reviews Molecular Cell Biology, 2015). These interactions are enabled by electrostatic forces due to the different chemical characteristics of the amino acids. Nowadays, many biochemical or microscopy-based methods are available to investigate protein-protein interactions, for instance protein complex immunoprecipitation (Co-IP), Förster resonance energy transfer (FRET) or yeast two-hybrid screenings. As easy it is to detect a protein-protein interaction, as difficult it is to alter the strength of these interactions, although the magnitude of interaction can influence the cellular outcome, for example a stronger signal transduction or a higher expression rate. Phage-assisted continuous evolution (PACE) allows a directed evolution of different kinds of proteins within a few days as it was for example shown for RNA polymerases, proteases or aminoacyl-tRNA synthetases. (Esvelt et al, Nature, 2011; Dickinson et al, Nat Commui., 2014; Bryson et al, Nat Chem Biol. 2017). Technically, a directed evolution towards a tighter protein-protein interaction is possible as well, as it was shown for the toxin Cry1Ac from Bacillus thuringiensis (Bt toxin) that binds a cadherin-like receptor (Badran et al, Nature, 2016).Motivation
A special type of a protein-protein interaction are split enzymes that allow an auto-reassembly. They usually consist of two enzyme fragments (an N‑terminal and C‑terminal fragment) that are expressed separately, and fuse post translation. This protein fragment complementation is advantageous for any kind of application where the size of an expression cassette is a limiting factor, for instance for the packaging of DNA into virus capsids.For many enzymes auto-reassembly split sites are known, however the efficiency of the joined fragments usually does not reach wildtype efficiency, as it was shown for Cas9 enzymes (Zetsche et al, Nature Biotechnology, 2015; Kaya et al, Plant Cell Physiol, 2017) Consequently, a directed evolution of split enzymes that enhance auto-reassembly is of great interest for the synthetic biology community. To demonstrate that an improved protein-protein interaction is not only possible for the Bt toxin, we used a split T7-polymerase as another example (Tiun Han et al, ACS Synthetic Biolog, 2017). We aimed at enhancing the auto-reassembly efficiency of different split sites using two in our lab established methods, PACE and PREDCEL (phage-related discontinuous evolution), which allow a fast and relatively easy directed evolution.
Figure One: Selecting for efficient T7-RNAP auto-reassembly in directed evolution experiment
As all PREDCEL/PACE experiments are based on phage infection, selection for efficient T7-RNAP auto-reassembly starts with a phage injecting its genome, the selection plasmid (SP), into a bacterial cell. The SP carries the two halves of the split T7 RNAP expressed under one geneIII (gIII) promotor. Further it contains required genes for phage reproduction except for gIII.
If through MP-activation mutation process leads to beneficial mutations in one or both T7 RNAP sequences, split variants assemble efficiently, bind to T7 promotor and thereby activate expression of protein III (pIII). Now phages can be built including pIII. That enables the phage to be released and to infect new cells (left).
If mutations are inefficient, T7 RNAP halves do not reassemble, therefore cannot bind to T7 promotor and pIII is not expressed. Therefore, phages cannot leave cells or infect new cells (right).
